# Best practices for custom sequencing primer use on the NextSeq 1000/2000
Custom sequencing primers are enabled with NextSeq 1000/2000 Control Software v1.2+.
Refer to the [NextSeq 1000/2000 custom primers guide](https://support-docs.illumina.com/IN/NextSeq10002000/Content/IN/NextSeq2000_1000/PrepareAddCP_intro_fNS_m2000.htm) for guidance on preparing and adding custom primers to the reagent cartridge, including volumes and concentrations to use.
**Best practices**
* **In most cases Illumina recommends combining custom Read 1 and Read 2 primers in one well, and custom Index 1 and Index 2 primers in the other well.** NextSeq 1000/2000 Custom Primers Guide and NextSeq 1000/2000 Control Software allow any combination of sequencing primers in one well however, keep in mind the following (relevant to **most** library types):
* Read 1 and i7 index (index 1) are sequenced on the same strand and Read 2 and i5 index (index 2) are also sequenced on the same strand.
* Read 1 and i5 index (index 2) and Read 2 and i7 index (index 1) are the reverse-complement of each other and will bind to each other.
* For reference, see figure 1 below.
**Figure 1:** The NextSeq 1000/2000 Reverse Complement Workflow Index Sequencing Scheme shows the binding region of read and index primers
* Combine no more than 2 custom primers per well. See Figured 2 below.
**Figure 2:** Reagent Cartridge layout detailing the location of custom primer wells (Well 1 and Well 2).
**PhiX Considerations**
PhiX requires the Illumina sequencing primers for proper priming and sequencing. When custom primers are selected, the standard Illumina sequencing primers are **not used** for the read or index priming during the sequencing run. If Illumina primers are not used for Read 1 or Read 2, the optional PhiX control is not primed and will not be sequenced.
* If the library pool includes optional PhiX spike-in or requires Illumina primers combined with custom sequencing primers, an additional NextSeq 1000/2000 primer kit must be purchased and added to the custom primer wells.
| **Kit name** | **Catalog#** |
| --------------------------------------------------- | ------------ |
| Standard SBS NextSeq 1000/2000 Read & Index Primers | 20046115 |
| Standard SBS NextSeq 1000/2000 Index Primer Kit | 20046116 |
| Standard SBS NextSeq 1000/2000 Read Primer Kit | 20046117 |
| XLEAP-SBS NextSeq 1000/2000 Read & Index Primer | 20112856 |
| XLEAP-SBS NextSeq 1000/2000 Index Primer Kit | 20112858 |
| XLEAP-SBS NextSeq 1000/2000 Read Primer Kit | 20112859 |
**How to designate Custom Primers in Run Setup**
Custom primers are compatible with Cloud, Hybrid, Local, and Standalone run modes. Indicate in the control software run setup page if a read requires a custom primer. Define which well (see Figure 3) for each read.
* For Cloud, Hybrid, or Local modes, custom primer selection takes place in the NextSeq 1000/2000 Control Software on the **Review** screen and is not setup in BaseSpace Run Planning or on the Sample Sheet.
* For Standalone Mode, Custom Primers are selected on the **Run Setup** screen.
**Figure 3:** Run Setup overview for custom primer selection.
Custom primer selections are indicated in the runparameters.xml file.
**Table 1:** Comparison of RunParameters.xml entries with and without custom primer usage.
| Custom Primer in Read 1 alone | No Custom Primer Used |
| -------------------------------------------------------- | --------------------------------------------------------- |
|  |  |
Custom primer recipes are available for the v2 P2 cartridge. These can be found on the [NextSeq 1000/2000 Sequencing System Product Files](https://support.illumina.com/sequencing/sequencing_instruments/nextseq-1000-2000/product-files.html) page under NextSeq 1000/2000 P2 (v2) Reagents Kit Custom Recipes for Custom Primers.
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2431), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002431%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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